Oral composition

ABSTRACT

An oral composition having a biofilm formation inhibitory action is provided. An oral composition, comprising a plant extract which is a hot water extract of Japanese mustard spinach, potherb mustard, mibuna, hot radish, cress, ostrich fern, Japanese angelica tree, or ice plant.

TECHNICAL FIELD

The present invention relates to an oral composition having aninhibitory action by a plant extract on Actinomyces naeslundii(hereinafter referred to as “A. naeslundii”) biofilm formation.

BACKGROUND ART

Microorganisms attached on the surface of an object do not existindependently, but form a biofilm together with other microorganisms inthe characteristic structure. The biofilm, which appears to work infavor of the human as seen in the use of immobilized microorganisms, isconversely revealed to cause tooth decay and food contamination, and hasbeen extensively studied in recent years.

The dental biofilm includes 700 species or more of bacteria and 10⁸ ormore bacteria are present per 1 mg of the dental biofilm. Of these, A.naeslundii has been receiving much attention in recent years as a keymicroorganism required when the initial stage plaque (cariogenic plaque)proceeds to the late stage plaque (periodontal disease plaque).

In the conventional periodontal disease prevention, the mainstream ideawas to sterilize periodontal disease bacteria such as Porphyromonasgingivalis (hereinafter referred to as “P. gingivalis”) to inhibit theperiodontal diseases. However, in reality, the periodontal disease fociare present with a biofilm deeply in the periodontal pocket, whichprevents an antibacterial substance from permeation. Therefore, theconventional periodontal disease prevention fails to achieve an intendedeffect, and additionally always facing the risk of the resistantbacteria emergence. Thus, a safer and more effective prevention methodhas been in demand.

Also, mechanical removal such as brushing, and scaling, is believed tobe the most effective method for inhibiting a biofilm, but it isdifficult to practice suitable oral care using the current procedure forpeople having difficulties in such a mechanical control of dentalbiofilm such as the elderly in need of nursing care, whereby thedevelopment of biofilm removal procedures different from the conventionprocedures are in demand.

Short chain fatty acids (SCFA) such as butyric acid, are known to bepresent in a high concentration in the periodontal pocket and the dentalplaque of periodontal disease patients, and the association thereof withthe development and progress of the periodontal diseases became clear.In recent years, it was confirmed that acids such as SCFA produced bythe periodontal disease bacteria such as P. gingivalis has a promotingeffect on biofilm formation of A. naeslundii, and the studies oninvestigation of mechanism and pathogenic development control whichtargets this phenomenon are conducted (NIHON UNIVERSITY SCHOOL OFDENTISTRY, National Institute of Infectious Diseases, Japan).

In the present invention, with an attention on the influence by acids tothe A. naeslundii biofilm formation, a substance, which is capable ofinhibiting the biofilm formation depending on acids such as butyric acidthereby to inhibit the periodontal disease biofilm formation, wassearched for, and a development of a novel periodontal disease biofilminhibiting material with such substance was attempted.

An example of the literature regarding an oral composition exhibiting anantibacterial effect on the dental biofilm and an improvement effect onthe gingivitis is PTL 1. PTL 1 discloses an oral composition containing(A) N-acylsarcosine or a salt thereof and (B) benzylisothiocyanate in amass ratio (A)/(B) of 0.5 to 20, and also discloses that the oralcomposition has a good antibacterial effect on the dental biofilmcausing the oral diseases and improvement effect on the gingivitis.However, PTL 1 does not disclose nor suggest any effects on theperiodontal diseases.

CITATION LIST Patent Literature

[0009]PTL 1: Japanese Patent Application Laid-Open No. 2008-174542

SUMMARY OF INVENTION Technical Problem

Conventionally, in the methods for inhibiting the periodontal diseaseswhich employ a certain antibacterial agent, the dental biofilm preventsthe permeation of antibacterial agents and thus makes it difficult toachieve the intended periodontal disease inhibitory effects. Further,the use of antibacterial agents involves a high risk of resistantbacteria emergence, hence is not preferable. For these reason,controlling biofilm formed by A. naeslundii, which related to theinitial stage of periodontitis is considered to be a periodontal diseaseprevention method which is safer and more effective than sterilizing theperiodontal disease bacteria.

Solution to Problem

The present inventors conducted extensive studies and found that each ofthe plant extracts of potherb mustard, Japanese mustard spinach, hotradish and peppergrass belonging to the Brassicaceae family, Japaneseangelica tree belonging to the Araliaceae family, and ice plantbelonging to the Aizoaceae family has an inhibitory effect on the A.naeslundii biofilm formation induced by acid, whereby the presentinvention was accomplished.

The biofilm formation of A. naeslundii increases by the acidstimulation, but when 1000 ppm of the above plant is added to theculture system, the biofilm formation was found to be reduced to about30%. At this time, some plant extracts did not affect the growth of A.naeslundii, suggesting that this biofilm formation inhibitory effect hasa different mechanism from the antibacterial action.

A. naeslundii is a Gram-positive bacillus found in the sites ofgingivitis and root surface caries and believed to be the pathogenicbacterium of the initial periodontal disease. Since A. naeslundiicoaggregates with Streptococcus and the periodontal disease bacteria, itis considered to be a bacterium which is the key to the transition ofbacterial flora to the periodontal plaque, which suggests that thecontrol of A. naeslundii leads to the effective periodontal diseaseprevention.

The present inventors have validated that the biofilm formation wasincreased by the acids produced by the oral bacteria.

ADVANTAGEOUS EFFECTS OF INVENTION

The present invention provides a periodontal disease preventive agentwith a new perspective, and is believed, when compared with the existingantibacterial agents, to have a lower risk of resistant bacteriaemergence, and the like, hence advantageous.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the A. naeslundii biofilm formation by theacid addition.

FIG. 2 is a graph showing the difference in the biofilm formationinhibitory activity by the extractant of a hot radish.

DESCRIPTION OF EMBODIMENTS

The present invention is directed to prevent the periodontal diseases byinhibiting the periodontal disease biofilm formed by A. naeslundii, aninitial stage periodontal disease bacterium, and based on the findingssuch a periodontal disease biofilm inhibitor in the extracts of a partof the Brassicaceae family plants such as potherb mustard, Japanesemustard spinach and hot radish; Japanese angelica tree; and ice plant.

Being unlike the conventional antibacterial agents used for periodontaldisease bacteria, the present inhibitor inhibits the biofilm formation,which is the dwelling of periodontal disease bacteria, and is believedto be effective and safe.

The present inhibitor is also applicable to chewing gums and candies asa novel oral composition. More specifically, the present inventionrelates to an oral composition containing a plant extract, wherein theplant extract is a hot water extract of Japanese mustard spinach,potherb mustard, mibuna (Brassica campestris var, laciniifolia), hotradish, cress, ostrich fern, Japanese angelica tree, or ice plant.

Further, the present invention relates to an acid-induced biofilmformation inhibitor containing a plant extract, wherein the plantextract is a hot water extract of Japanese mustard spinach, potherbmustard, mibuna, hot radish, cress, ostrich fern, Japanese angelicatree, or ice plant.

Furthermore, the present invention relates to mouthwashes, toothpastes,inhalants, troches, and food products such as chewing gums, candies, andtablets, comprising an oral composition containing a plant extract,which is a hot water extract of Japanese mustard spinach, potherbmustard, mibuna, hot radish, cress, ostrich fern, Japanese angelicatree, or ice plant.

EXAMPLE Example 1

(Extract Preparation)

Each of the plant samples was a commercially purchased product. Afterfreeze-dried, 1 g of each of the plant samples was finely ground using agrinder, and extracted with 50 ml of water at 70° C. for 2 hours. Theobtained extract was centrifuged at 3000 rpm for 10 minutes, thesupernatant thereof was filtered and freeze-dried to be tested as a hotwater extract of each plant.

Example 2

(Evaluation on the Biofilm Formation Inhibitory Activity)

1. Biofilm Formation

A. naeslundii ATCC19039 strain was anaerobically cultured in 5 ml ofBrain Heart Infusion (BHI) liquid media at 37° C. for 10 hours. Then,the bacteria collected by the centrifugal separation at 3000 rpm for 10minutes was treated with PBS to adjust to OD_(550 nm)=0.5 and used as atest sample suspension.

The test of biofilm formation was carried out using a 96-wellmicroplate. In each well, 60 μl of the biofilm formation inhibitorysample, 20 μl of butyric acid, 20 μl of a test sample suspensioncontaining A. naeslundiiand 100 μl of Toripticase soy broth with 0.5%sucrose added thereto were added and incubated for 16 hours at 37° C.under the conditions of 5% CO₂.

2. Quantitative Determination of Biofilm

The supernatant after the above incubation was removed and each well waswashed twice with PBS. After washing, a 0.25% safranine solution wasadded to each well, allowed to stand for 15 minutes. The excesssafranine solution was removed from each well and each well was washedtwice with PBS. After washing, ethanol was added to each well, thestained safranine was eluted with shaking for 30 minutes and theabsorbance at 492 nm was measured using a microplate reader to determinethe amount of formed biofilm.

The amount of formed biofilm was shown in terms of the percentage, theamount formed with no sample added being 100.

(Result)

1. Biofilm Formation Inhibitory Effect of Natural Product Extract

Extracts of various natural products were examined for the biofilmformation inhibitory activity in a butyric acid added system. Hot waterextracts of various plants were prepared and measured for the biofilmformation inhibitory activities, and it was confirmed that when the hotwater extract of Japanese mustard spinach, potherb mustard, mibuna, hotradish, cress, ostrich fern, Japanese angelica tree, or ice plant wasadded to the test system, the biofilm formation amount remainedsubstantially unchanged in the system to which butyric acid was notadded, whereas the biofilm formation amount was reduced by 50 to 90% inthe system to which butyric acid was added. The effect was outstandingparticularly in the hot water extracts of plants of the Brassicaceaefamily (Table 1).

Subsequently, the A. naeslundii biofilm formation was investigated forany increase by the addition of acids other than butyric acid, and apotherb mustard extract was investigated for the acid-induced biofilmformation inhibition. As a result, it is revealed that the A. naeslundiibiofilm formation was increased even with the addition of other acidsthan butyric acid, such as acetic acid, lactic acid, and hydrochloricacid, and the potherb mustard extract inhibits the biofilms thereof(FIG. 1).

Hot radish whose activity was validated was extracted using differentextractants (hot water, 50% ethanol, 100% ethanol), and each of whichwas examined for the biofilm formation inhibitory activity, and it wasfound that all the extract had the concentration dependent biofilmformation inhibitory activity (FIG. 2). These results suggest that thehot radish is most likely to contain both water soluble active substanceand lipid soluble active substance.

To develop a periodontal disease preventive material which works by amechanism different from the conventional materials, a butyricacid-induced biofilm formation inhibitory substance was developedfocusing on the phenomenon in which the A. naeslundii biofilm formationwas increased by butyric acid.

On the premise that the material is used for food products, the searchwas carried out mainly in the plants commonly eaten and remarkableactivities were found in several kinds of plant extracts. These extractsdid not notably inhibit the biofilm formation in the system to whichbutyric acid was not added but inhibited in the system to which butyricacid was added, indicating that the extracts were likely to affect onlythe system associated with butyric acid. In the present tests, theactivity was found in the Brassicaceae family plants. But some of theBrassicaceae family plants do not exhibit the biofilm formationinhibitory activity. So, the effect was not common in all of theBrassicaceae family plants.

Also, the significant biofilm formation inhibitory activity was found inthe extract of ice plant, an Aizoaceae family plant. Ice plant(scientific name: Mesembryanthemum crystallinum) is a salinity toleranthalophilous plant, native to Europe, western Asia and Africa, and can behydroponically cultivated in a sodium chloride aqueous solution having asaline concentration substantially equivalent to the seawater. Ice plantis sold at the vegetable section of department stores and grocery storesin recent years and has been receiving attention. If additional valuessuch as the periodontal disease biofilm prevention effect, were found,even more attention will be paid thereto.

The control of microorganisms and the conquer of infectious diseasesusing antibacterial agents, germicides, or the like, are the challengesagainst the resistant bacteria emergence, and the endless battlesbetween novel antibiotics development and resistant bacteria emergencehave been still ongoing at present. However, the biofilm formationinhibitory activity found in the plant hot water extracts obtained thistime is verified only at the time of adding acids, and therefore, it islikely to be very safe in view of the resistant bacteria emergence.

Based on the results in this invention, investigation of the mechanismof the effect, identification of the active ingredient and effect testson human will be carried out hopefully to develop a novel periodontaldisease biofilm formation inhibitory material and apply such a materialto oral care products.

Substances inhibiting the A. naeslundii biofilm formation induced by theaddition of butyric acid were searched and it was found that the hotwater extracts of 5 varieties belonging to the Brassicaceae plants suchas Japanese mustard spinach and potherb mustard, and ice plant inhibitedthe biofilm formation by 50 to 90%. Further, it was suggested that theactive ingredient of the Brassicaceae plants contains both of watersoluble and lipid soluble substances.

TABLE 1 Biofilm formation inhibitory effect of hot water extracts ofnatural products Sample Sample 500 ppm 1000 ppm Butyric acid Butyricacid Sample Family Genus − + − + — 100.0 100.0 100.0 100.0 1 BeetChenopodiaceae Beta 87.3 104.5 132.0 96.4 2 Swiss chard ChenopodiaceaeBeta 333.6 80.2 327.8 30.6 3 Raw coffee bean Rubiaceae Coffea 84.5 16.819.0 12.0 4 Coffee bean Rubiaceae Coffea 260.9 35.6 4.8 22.3 5 Indianmulberry Rubiaceae Morinda 231.3 93.6 584.8 92.3 6 Japanese mustardspinach Brassicaceae Brassica 537.7 93.2 87.9 6.8 7 Potherb mustardBrassicaceae Brassica 113.1 8.0 116.7 8.9 8 Mibuna Brassicaceae Brassica94.1 13.2 138.1 33.8 9 Turnip Brassicaceae Brassica 455.8 77.7 367.071.4 10 Broccoli Brassicaceae Brassica 51.8 101.4 68.9 98.8 11 Rapeblossom Brassicaceae Brassica 297.4 101.8 278.2 104.5 12 Kakina leafBrassicaceae Brassica 156.7 115.4 325.7 115.8 13 Wasabi leafBrassicaceae Brassica 86.7 110.9 45.6 118.9 14 Watercress BrassicaceaeNasturtium 44.2 50.9 7.9 26.8 15 Rucola Brassicaceae Arugula 11.8 30.17.8 38.0 16 Red radish Brassicaceae Raphanus 50.9 22.7 39.2 11.2 17 Hotradish Brassicaceae Raphanus 91.0 83.2 81.9 27.5 18 Radish BrassicaceaeRaphanus 49.2 97.2 46.6 74.6 19 Cress Brassicaceae Lepidium 523.1 87.7302.2 36.9 20 Horse radish Brassicaceae Wasabia 80.4 71.7 57.6 73.0 21Ostrich fern Woodsiaceae Matteuccia 29.7 23.4 20.8 11.5 22 Japaneseangelica tree Araliaceae Aralia 117.0 9.5 103.9 1.8 23 ZucchiniCucurbitaceae Cucurbita 337.9 86.4 329.9 91.9 24 Pumpkin CucurbitaceaeCucurbita 63.3 129.7 70.1 140.6 25 Cucumber Cucurbitaceae Cucumis 316.382.7 313.3 82.6 26 Melon cucumber Cucurbitaceae Cucumis 291.8 87.8 327.390.0 27 Okinawan yellow cucumber Cucurbitaceae Cucumis 107.1 124.5 97.5117.1 28 Bitter melon Cucurbitaceae Momordica 522.6 77.5 568.1 79.8 29Wax gourd Cucurbitaceae Benincasa 134.5 96.7 117.1 95.3 30 ChicoryAsteraceae Cichorium 175.8 103.7 315.3 103.6 31 Gynura bicolorAsteraceae Gynura 145.0 100.4 137.6 105.7 32 Nigana Asteraceae Ixeris94.7 91.1 87.4 95.4 33 Butterbur scape Asteraceae Petasites 509.7 124.9511.2 129.3 34 W blueberry Ericaceae Vaccinium 168.6 98.8 212.8 102.0 35Chinese wolfberry fruit Solanaceae Lycium 187.7 87.6 476.1 84.6 36 Sweetgreen pepper Solanaceae Capsicum 137.1 73.6 544.3 64.8 37 Manganjipepper Solanaceae Capsicum 102.7 81.0 118.5 82.0 38 Green pepperSolanaceae Capsicum 129.9 91.4 221.1 83.7 39 Green chili SolanaceaeCapsicum 64.9 87.3 37.3 84.1 40 Jalapeno pepper Solanaceae Capsicum103.7 98.9 72.7 85.9 41 Eggplant Solanaceae Solanum 235.5 70.2 579.366.1 42 Potato Solanaceae Solanum 166.2 95.7 229.8 97.2 43 Cherry tomatoSolanaceae Solanum 193.8 102.9 372.1 102.9 44 Mizunasu eggplantSolanaceae Solanum 127.7 117.0 151.7 108.3 45 Tomato Solanaceae Solanum164.4 103.1 131.4 110.7 46 Beinasu eggplant Solanaceae Solanum 217.5106.2 259.0 111.4 47 Pepino Solanaceae Solanum 93.7 81.7 109.8 55.4 48Red onion Alliaceae Allium 447.4 111.3 660.6 96.0 49 Ice plant AizoaceaeMesembryanthemum 504.8 34.2 213.7 11.0 50 Prune Rosaceae Prunus 263.8107.6 401.7 66.0 51 Apricot Rosaceae Prunus 126.0 108.6 388.5 112.7 52Cherry Rosaceae Prunus 313.5 124.3 485.0 183.3 53 Kidney bean FabaceaePhaseolus 240.3 86.9 320.0 86.5 54 Red kidney bean Fabaceae Phaseolus66.2 122.8 55.7 103.3 55 Feijoada bean Fabaceae Phaseolus 82.3 120.8106.5 103.6 56 Black kidney bean Fabaceae Phaseolus 122.8 104.2 162.7115.1 57 White flower bean Fabaceae Phaseolus 102.4 116.4 84.8 122.3 58Red pea Fabaceae Pisum 182.3 123.1 275.5 121.6 59 Mung bean FabaceaeVigna 70.6 95.5 45.9 37.9 60 Dainagon azuki Fabaceae Vigna 87.1 96.569.5 48.2 bean 61 Bean sprout Fabaceae Vigna 99.5 97.3 106.1 100.8 62Black-eyed pea Fabaceae Vigna 132.2 110.9 103.3 114.2 63 Winged beanFabaceae Psophocarpus 72.1 89.0 131.4 88.4 64 Lentil bean Fabaceae Lens244.9 120.4 457.2 115.8 65 Asparagus Liliaceae Asparagus 106.1 97.1150.3 100.0 66 Dogtooth violet Liliaceae Erythronium 168.7 81.7 240.765.7 67 Hosta montana Liliaceae Hosta 229.7 100.0 325.4 98.2

Subsequently, a mouthwash, toothpaste, breath freshening spray, troche,chewing gum, candy, tablet candy, gummy jelly, and beverage, allcontaining the acid-induced biofilm formation inhibitor containing theplant extract of the present invention, were produced by a conventionalmethod. The formulae are shown below.

However, the scope of the present invention is not limited thereto.

Example 3

A mouthwash was produced according to the following formulation.

Ethanol 2.0% by weight Japanese mustard spinach extract 1.0 Flavor 1.0Water Balance 100.0

Example 4

A toothpaste was produced according to the following formulation.

Calcium carbonate 50.0% by weight Glycerol 19.0 Mibuna extract 1.0Carboxymethylcellulose 2.0 Sodium lauryl sulfate 2.0 Flavor 1.0Saccharin 0.1 Chlorhexidine 0.01 Water Balance 100.0

Example 5

A breath freshening spray was produced according to the followingformulation.

Ethanol 10.0% by weight Glycerol 5.0 Hot radish extract 1.0 Flavor 0.05Coloring agent 0.001 Water Balance 100.0

Example 6

A troche was produced according to the following formulation.

Cress extract 92.3% by weight Gum Arabic 6.0 Flavor 1.0 Sodiummonofluorophosphate 0.7 100.0

Example 7

A chewing gum was produced according to the following formulation.

Gum base 20.0% by weight Xylitol 54.7 Maltose 15.0 Sorbitol 9.3 Flavor0.5 Potherb mustard extract 0.5 100.0

Example 8

A candy was produced according to the following formulation.

Sugar 50.0% by weight Reduced sugar syrup 33.0 Citric acid 1.0 Flavor0.2 L-Menthol 1.0 Potherb mustard extract 0.4 Water Balance 100.0

Example 9

A tablet candy was produced according to the following formulation.

Sugar 74.7% by weight Lactose 18.9 Ice plant extract 2.0 Fatty acidsucrose ester 0.15 Water 4.25 100.0

Example 10

A gummy jelly was produced according to the following formulation.

Gelatin 60.0% by weight Reduced sugar syrup 32.4 Japanese angelica treeextract 0.5 Vegetable oil and fat 4.5 Malic acid 2.0 Flavor 0.5 100.0

Example 11

A beverage was produced according to the following formulation.

Orange juice 30.0% by weight Ostrich fern extract 0.5 Citric acid 0.1Vitamin C 0.04 Flavor 0.1 Water Balance 100.0

INDUSTRIAL APPLICABILITY

The oral composition of the present invention is a periodontal diseasepreventive agent with a new perspective which exhibits the acid-inducedperiodontal disease biofilm formation inhibitory action, different fromthe conventional antibacterial agents against periodontal diseasebacteria. Consequently, the oral composition of the present invention islikely to render advantages of having a lower risk of resistant bacteriaemergence, or the like, when compared with the existing antibacterialagents, and the like, and thus applicable to various products.

This application claims the priority from Japanese Patent ApplicationNo. 2011-196315, filed on Sep. 8, 2011, and the disclosure of which ishereby incorporated by reference as a part of the present application.

1. An oral composition, comprising a plant extract.
 2. The oralcomposition according to claim 1, wherein the plant extract is a hotwater extract of Japanese mustard spinach, potherb mustard, mibuna, hotradish, cress, ostrich fern, Japanese angelica tree, or ice plant.
 3. Anacid-induced biofilm formation inhibitor, comprising a plant extract. 4.The acid-induced biofilm formation inhibitor according to claim 3,wherein the plant extract is a hot water extract of Japanese mustardspinach, potherb mustard, mibuna, hot radish, cress, ostrich fern,Japanese angelica tree, or ice plant.
 5. A mouthwash comprising the oralcomposition according to claim
 1. 6. A toothpaste comprising the oralcomposition according to claim
 1. 7. An inhalant comprising the oralcomposition according to claim
 1. 8. A troche comprising the oral oralcomposition according to claim
 1. 9. A food product containing the oralcomposition according to claim
 1. 10. A mouthwash comprising the oralcomposition according to claim
 2. 11. A toothpaste comprising the oralcomposition according to claim
 2. 12. An inhalant comprising the oralcomposition according to claim
 2. 13. A troche comprising the oralcomposition according to claim
 2. 14. A food product containing the oralcomposition according to claim 2.